It is desirable to identify useful methods of purifying proteins that do not destroy, or significantly reduce, the biological activity of the protein. Contaminants must be removed from protein preparations, such as acidic protein preparations (e.g., immunoglobulin (Ig)-fusion protein preparations), before they can be used in diagnostic applications, therapeutic applications, applied cell biology, and functional studies. For instance, protein preparations, e.g., acidic protein preparations, often contain unwanted components (impurities), such as inactive and/or partially active species and high molecular weight aggregates (HMWA). Presence of inactive and/or partially active species is undesirable because these species have significantly lower binding capacity to the target compared to the active protein; thus, the presence of inactive and/or partially active species can reduce product efficacy. The formation of aggregates, e.g., HMWA, can adversely affect product safety by causing complement activation or anaphylaxis upon administration. Further, aggregate formation may hinder manufacturing processes by causing decreased product yield, peak broadening, and loss of activity.
The most common protein purification methods are predicated on differences in the size, charge, and solubility between the protein to be purified and contaminants. Protocols based on these parameters include affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. These chromatographic methods, however, sometimes present technical difficulties in the separation of aggregated or multimeric species of proteins, e.g., IgG-containing proteins. Techniques such as ion exchange and hydrophobic interaction chromatography, for instance, may induce the formation of aggregates due to an increased protein concentration or the required changes in buffer concentration and/or pH during elution. Further, in several instances proteins show differences in isoelectric points that are too small to allow for their separation by ion-exchange chromatography. Tarditi, (1992) J. Immunol. Methods 599:13-20. Size exclusion chromatography is cumbersome and results in the significant dilution of the product, which is a hindrance in large-scale, efficiency-based manufacturing processes. Leakage of ligands from affinity chromatography columns can also occur, which results in undesirable contamination of the eluted product. Steindl (2000) J. Immunol. Methods 235:61-69. Of interest, Applicants were unable to remove the inactive or partially active species using either ion exchange, e.g., anion exchange, or hydrophobic interaction chromatography.
Hydroxyapatite chromatography is a method of purifying proteins that utilizes an insoluble hydroxylated calcium phosphate [Ca10(PO4)6(OH)2], which forms both the matrix and ligand. Functional groups consist of pairs of positively charged calcium ions (C-sites) and clusters of negatively charged phosphate groups (P-sites). The interactions between hydroxyapatite and proteins are complex and multi-mode. In one method of interaction, positively charged amino groups on proteins associate with the negatively charged P-sites, and protein carboxyl groups interact by coordination complexation to C-sites. Shepard (2000) J. of Chromatography 891:93-98. Thus, acidic and basic proteins usually interact with cHA resin through different mechanisms: an acidic protein usually binds to C-sites via a coordination bond to carboxyl group, while a basic protein binds to P-sites through charge interaction with the amine group. Crystalline hydroxyapatite was the first type of hydroxyapatite used in chromatography, but it was limited by structural difficulties. Ceramic hydroxyapatite (cHA) chromatography was developed to overcome some of the difficulties associated with crystalline hydroxyapatite, such as limited flow rates. Ceramic hydroxyapatite has high durability, good protein binding capacity, and can be used at higher flow rates and pressures than crystalline hydroxyapatite. Vola et al. (1993) BioTechniques 14:650-655. Chromatographic separation using cHA can be performed in several distinct modes, such as binding mode, flow-through mode, or a combination binding/flow-through mode.
Hydroxyapatite chromatography has been used in the chromatographic separation of proteins, nucleic acids, as well as antibodies. However, in several instances, researchers have been unable to selectively elute antibodies from hydroxyapatite or found that hydroxyapatite chromatography did not result in a sufficiently pure product. Junbauer, (1989) J. Chromatography 476:257-268; Giovannini, (2000) Biotechnology and Bioengineering 73:522-529. A successful separation of antibodies and other basic proteins from impurities, such as HMWA, using cHA chromatography either in binding, flow-through, or combination binding/flow-through mode has been demonstrated in U.S. Publication No. 2005-0107594, incorporated herein in its entirety by reference. The present invention provides a novel method for removing product-related partially active and/or inactive species, as well as other impurities, such as HMWA, from acidic proteins, e.g., Ig-fusion proteins, using cHA chromatography techniques.